Functional Analysis Of Arabidopsis HOPs And SIL In Response To Abiotic Stresses

1 HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex.Arabidopsis heat shock protein(HSP)70-HSP90 organizing protein(HOP)is a cochaperone of HSP90.There are three members in HOP gene family,HOP1,HOP2 and HOP3.HOPs play an important role in thermo tolerance of plants.The hop 1/2 had salt-sensitive phenotype.In this study,we discovered the molecular mechanism that HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid(BR)-related nucleo-cytoplasmic partitioning of the HSP90-brassinosteroid-insensitive 2(BIN2)complex.The main results were shown as follows:1.1 The salt-sensitivity of hop1/2 was similar to that of hop1/2/3,and the restoration of HOP2 expression remedied the salt-sensitive defect of hop1/2,and two independent mutants,hop1-1 and hopl-2,showed no salt-sensitive phenotype,indicating that HOP1 and HOP2 are involved in salt tolerance,and there was functional redundancy between HOP1 and HOP2.1.2 hop1/2 mutant showed no sensitive to mannitol and LiCl stresses,indicating that the increased salt-sensitivity of hop1/2 was not caused by ionic toxicity or osmotic stress.In addition,the Na+ and K+contents of hop1/2 were the same as WT during NaCl stress,indicating that the increased Na+ content did not account for the increased salt-sensitivity of hop1/2.1.3 The endogenous BR content was obviously lower in hop1/2 than that in WT during NaCl stress,exogenous brassinolide(BL)effectively remedied the salt-sensitivity of hop1/2 and brassinazole(BRZ)also eliminated the differential growth between WT and hop1/2 under salt stress.These results indicated that under salt stress,the low concentration of endogenous BR is responsible for the slow growth of hop1/2.1.4 Under NaCl stress,mutation of both HOP1 and HOP2 impeded the phosphorylation and dephosphorylation of brassinazole-resistant 1(BZR1)and affected BR-related gene expression.Therefore,under salt-stress,the mutation of HOP1 and HOP2 abrupted the synthesis of endogenous BR,thus preventing BR response.1.5 HOP1 and HOP2 interact with HSP90 and BIN2,a negative regulator in the BR signaling pathway.Under salt-stress,HOP1,HOP2,HSP90 and BIN2 accumulated in the nucleus of WT,while HSP90 and BIN2 contents in the nucleus did not significantly change in hop1/2.Therefore,under salt-stress,the low concentration of endogenous BR in hop1/2 failed to promote the growth of plant.As a result,hop1/2 was sensitive to salt-stress.In brief,we found that hop1/2 showed increased sensitivity to NaCl which was remedied by the exogenous BL application.Knockouts of both HOP1 and HOP2 led to,under NaCl-stress,BR deficiency and retarded hop1/2 growth.This study reveals a new mechanism of HOP 1/2 function in BR regulation of salinity tolerance.2 Arabidopsis SIL is involved in abiotic stresses.Arabidopsis FES1A is a co-chaperone of HSP70.fes1a presented no any visible defect during growth and development.In this study,an unknown protein in Arabidopsis,with its structure similar to FES1A,has been explored.Phylogenetic tree analysis shows that the FES1Alike gene,yeast SIL and human SIL come from a common ancestor.Yeast SIL and human SIL are endoplasmic reticulum(ER)proteins,thus we named the FES1A-like gene as the Arabidopsis SIL gene.We explored the function of Arabidopsis SIL.The main results were shown as follows:1.1 We generated SIL-GFP transgenic lines.The fluorescence signal of SIL-GFP was well in agreement with the red fluorescence signal distribution of ER-RFP,indicating that SIL is an endoplasmic reticulum protein.1.2 The expression pattern of SIL was analyzed with the SILpro-GUS transgenic lines.As a result,SIL mainly expressed in young leaves and was induced by ER-stress and heat stress.1.3 sil mutants were generated by performing the Cas9 method.The introduced mutation sites were located in the first exon of SIL gene.All sil mutants presented no any visible defect in their growth and development,but sil mutants had significantly increased resistance to tunicamycin and DTT stresses.The expression level of BIP3 was significantly higher in sil relative to WT under ER-stress conditions.SIL is suggested being a negative regulator of ERstress response.Interestingly,the ER-stress resistance of sil bzip28 double mutant was higher than that of bzip28,but sil bzip60 double mutant manifested its ER-stress sensitivity as same as the bzip60.Therefore,the negative regulation of SIL on ER-stress response may be through the bZIP60-related signalling pathway.1.4 sil mutants presented increased thermotolerance compared with WT.During the heat recovery period,the level of spliced bZIP60 mRNA was significantly higher in sil than WT,and so did the protein level of BIP.The increased ER-stress response in sil may be responsible for the improved thermotolerance of sil.In brief,Arabidopsis endoplasmic reticulum protein SIL is involved in molecular regulation of endoplasmic reticulum and high temperature stresses.The innovations of this study are:HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex in Arabidopsis.SIL is localized in ER and negatively regulates ER-stress response.SIL plays an important role in heat-stress response in Arabidopsis.